Separating particular materials from fluids that contain complex combinations of constituents has traditionally been a difficult and expensive task. The importance and feasibility of making such separations have increased to the point that a new branch of science, referred to as separation science, has been recognized. Polyanions are one class of materials for which satisfactory methods of separation are inadequate. For example, there is no adequate, biocompatible method of separating polyanions, such as heparin and certain blood coagulating protein factors like factor X, from blood or other fluids.
Heparin exists mainly in the lungs, intestine, and liver of a variety of mammals. Heparin is richly found intracellularly in mucosal mast cells, connective tissue mast cells and basophilic leukocytes. Commercial heparin preparations are mostly obtained from porcine intestinal mucosa or beef-lung. It is composed of alternating, 1-4 linked uronic acid and D-glucosamine. The uronic acid residues are either L-iduronic acid or D-glucuronic acid; D-Glucosamine residues are either N-sulfated (major proportion) or N-acetylated (minor proportion). Heparin is extremely heterogeneous in both structure and molecular weight because the biosynthesis of its precursors, heparinproteoglycans (M. W. 750,000 to 1,000,000), is usually not completed. Low molecular weight heparin refers to the fractionated or depolymerized heparin, which has a lower molecular weight than the normal commercial grade heparin.
The anticoagulant properties have been demonstrated to be associated with heparin binding to Antithrombin III (ATIII). ATIII is a plasma glycoprotein with molecular weight approximately 58,000. ATIII binds with thrombin very tightly at a 1:1 stoichiometric ratio, which blocks the active site on thrombin and prevents it from interacting with fibrinogen. However, the inhibition rate of thrombin with ATIII is low in absence of heparin. Heparin dramatically accelerates the rate of thrombin inactivation up to 2000-fold. Clinically used heparin can be separated into two distinct fractions according to its affinity for ATIII. Approximately 33% of heparin has a high affinity for ATIII, which has potent anticoagulant activity (up to 90% of the activity of the unfractionated heparin). A low-affinity heparin binds to the same site on ATIII, but with approximately 1000 times lower affinity.
Although anticoagulation is the major pharmacological activity, heparin has many other functions. Heparin inhibits the proliferation of vascular smooth muscle cells and renal mesengial cells, suppresses the delayed-type hypersensitivity, and inhibits angiogenesis. Other pharmacological functions of heparin include antithrombotic effect, antibacterial, antivirus, and antitumor angiogenesis, particularly in combination with cortisone. Although it has been clinically observed that heparin may induce thrombocytopenia, in vitro studies have shown that normal heparin enhances the release of platelets. Moreover, various heparin-binding growth factors can be purified with heparin affinity chromatography.
Heparin has been extensively used in many clinical applications, including cardiac surgery, peripheral vascular surgery, dialysis, autotransfusion, transplantation, the treatment of pulmonary embolism, disseminated intravascularcoagulation, and venous thrombosis. The dosage is dependent on the type of application. Heparin has also been used as a prophylactic agent against deep vein thrombosis. The dose of heparin for this treatment is relatively low, e.g., 10,000 U/24 hr for subcutaneous administration. Heparin is also of value in the treatment of thromboembolic disorders, such as pulmonary embolism and arterial thrombosis. These treatments require relatively high doses of heparin, approximately 30,000 U/24 hr.
As a polyanion, many properties and applications of heparin are associated with electrostatic interactions. Binding of negatively charged heparin onto polycationic surfaces has been applied in the biomedical field in two major ways.
One is heparin immobilized nonthrombogenic surfaces. As an anticoagulant, heparin has been fixed onto polymers with positive charges by forming a stable complex. The immobilized heparin on the surface is released into blood by ion exchange, subsequently, the released free heparin interacts with ATIII. Heparin was complexed with benzalkonium, bearing quaternary ammonium moiety, mixed with graphite, and developed as a graphite-benzalkonium chloride-heparin (GBH) surface in 1961. This surface showed a thrombogenic resistance, however, the heparin release rate was too high to be used in long term applications. Many other polycationic surfaces have been developed in order to ionically bind heparin strongly, thereby giving a lower heparin release rate. Although these heparin-immobilized biomaterials have shown an improved in vitro and in vivo hemocompatibility, there remains major unresolved problems, i.e. the high release rate of heparin and leakage of cationic reagents. To overcome these obstacles, polycations have been adapted to immobilize heparin. Poly(amido-amine) grafted 33polyurethane (PUPA), and polyvinylchloride grafted with both polyethyleneglycol monomethacrylate and quarternized dimethylaminoethyl methacrylate (Anthron) have shown good long term blood compatibility.
A second field of application relates to heparin neutralization. Excessive heparin in the blood can be attracted and thus removed by electrostatic interactions with polycationic surfaces.
From the above it is apparent that the same principle of heparin interaction with polycationic surfaces can be used for different purposes, i.e. to release heparin into the blood or remove heparin from the blood. In the case of heparinized nonthrombogenic surfaces, heparin electrolytically binds with the polycationic surface before contact with blood. When the heparinized surface is exposed to blood, the immobilized heparin undergoes sustained release from the surface. In contrast, in the case of the removal of heparin from blood, heparin is the blood at high concentrations before it contacts the polycationic surfaces. Heparin electrostatically binds with the polycationic surfaces after exposure to blood and thus is removed from the blood.
Because the high level of heparin for prolonged period of time is contraindicated, numerous efforts have been made to minimize the adverse effects of heparin in the blood. These approaches are diversified into at least three groups. One is the administration of protamine to neutralize the heparin effects. A second if the use of heparin derivatives as anticoagulants, such as low molecular weight heparin. A third is the minimization of the dose of heparin. Each one of these approaches has serious drawbacks.
Intravenous protamine administration often leads to adverse hemodynamic interactions and causes a sudden fall in blood pressure. In addition, cardiovascular suppression, system hypotension, pulmonary hypertension, anaphylaxis, and complement activation have also been reported after protamine administration. Moreover, a heparin rebound effect and consequent bleeding may occur hours after initial heparin neutralization by protamine. Therefore, the dose of protamine needs to be carefully chosen because insufficient neutralization may still induce hemorrhagic complications and overdose of protamine is also contraindicated. For these reasons the use of protamine for heparin neutralization is still very difficult.
The use of a low molecular weight heparin derivative is not as effective as an anticoagulant compared with high molecular weight heparin. Low molecular weight heparin loses its effectiveness more rapidly since it breaks down in the body more readily than high molecular weight heparin.
The administration of minimal doses of heparin is dangerous since this increases the likelihood of unwanted coagulation after surgery thus forming unwanted blood clots in the arteries and veins.
The level of heparin in whole blood and blood plasma both in vitro and in vivo is critical to the well-being of the patient. It has been a problem to remove heparin from whole blood and blood plasma. The present invention aids in the solution of these long-standing problems.
Factor X is a key blood clotting factor in human physiology. Blood coagulation occurs through a complex series of reactions known as the clotting or coagulation cascade. This cascade is regulated by a series of zymogen (inactive enzyme precursor) to enzyme conversions that ultimately results in polymerization of insoluble fibrin. This insoluble fibrin becomes cross-linked and, together with platelets and other components of the blood, forms a blood clot. There are two pathways, intrinsic and extrinsic, that make up the clotting cascade for blood coagulation. The intrinsic pathway is initiated by activation of blood factor XII while the extrinsic pathway is initiated by release of tissue thromboplastin after injury to blood vessels. The intrinsic and extrinsic pathways merge in a step of the cascade involving activation of factor X. Thus, factor X plays a key role in blood clotting because it is common to both blood clotting pathways and is the blood factor at the critical point where the two pathways join. After this merger of the two pathways, one series of reactions leads to formation of the insoluble fibrin.
Factor X is a protein that is synthesized in the liver and depends on vitamin K for its synthesis. An amino terminal domain of factor X contains several .gamma.-carboxyglutamic acid residues. Each of these modified glutamic acid residues contains an additional free carboxyl group, thus creating a highly negatively charged region in the protein. Thus, factor X is a polyanion and can be separated from fluids in a manner similar to that used for separating heparin.